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- PDB-6xmf: Cryo-EM structure of Cas12g binary complex -

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Basic information

Entry
Database: PDB / ID: 6xmf
TitleCryo-EM structure of Cas12g binary complex
Components
  • CRISPR-Cas
  • RNA (116-MER)
KeywordsHYDROLASE/RNA / CRISPR / HYDROLASE-RNA complex
Function / homologyRNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesmetagenome (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsChang, L. / Li, Z. / Zhang, H.
CitationJournal: Nat Chem Biol / Year: 2021
Title: Cryo-EM structure of the RNA-guided ribonuclease Cas12g.
Authors: Zhuang Li / Heng Zhang / Renjian Xiao / Ruijie Han / Leifu Chang /
Abstract: Cas12g, the type V-G CRISPR-Cas effector, is an RNA-guided ribonuclease that targets single-stranded RNA substrate. The CRISPR-Cas12g system offers a potential platform for transcriptome engineering ...Cas12g, the type V-G CRISPR-Cas effector, is an RNA-guided ribonuclease that targets single-stranded RNA substrate. The CRISPR-Cas12g system offers a potential platform for transcriptome engineering and diagnostic applications. We determined the structures of Cas12g-guide RNA complexes in the absence and presence of target RNA by cryo-EM to a resolution of 3.1 Å and 4.8 Å, respectively. Cas12g adopts a bilobed structure with miniature REC2 and Nuc domains, whereas the guide RNAs fold into a flipped 'F' shape, which is primarily recognized by the REC lobe. Target RNA and the CRISPR RNA (crRNA) guide form a duplex that inserts into the central cavity between the REC and NUC lobes, inducing conformational changes in both lobes to activate Cas12g. The structural insights would facilitate the development of Cas12g-based applications.
History
DepositionJun 30, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 13, 2021Provider: repository / Type: Initial release
Revision 1.0Jan 13, 2021Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jan 13, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jan 13, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Jan 13, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jan 13, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Jan 13, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jan 13, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Jan 13, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jan 13, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Jan 13, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jan 13, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Jan 13, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jan 13, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Jan 13, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jan 13, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Feb 3, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Feb 10, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Apr 7, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4May 10, 2023Group: Database references / Source and taxonomy
Category: database_2 / em_entity_assembly_naturalsource / entity_src_gen
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_entity_assembly_naturalsource.ncbi_tax_id / _em_entity_assembly_naturalsource.organism / _entity_src_gen.gene_src_details / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name
Revision 1.5Jun 7, 2023Group: Source and taxonomy / Structure summary / Category: em_entity_assembly / pdbx_entity_src_syn
Item: _em_entity_assembly.source / _pdbx_entity_src_syn.details ..._em_entity_assembly.source / _pdbx_entity_src_syn.details / _pdbx_entity_src_syn.ncbi_taxonomy_id / _pdbx_entity_src_syn.organism_scientific
Revision 1.6May 15, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond
Revision 1.7May 28, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details
Item: _em_admin.last_update / _em_software.name / _pdbx_entry_details.has_protein_modification
Revision 1.1May 28, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

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Assembly

Deposited unit
A: CRISPR-Cas
C: RNA (116-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)129,3143
Polymers129,2482
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area8810 Å2
ΔGint-48 kcal/mol
Surface area45070 Å2

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Components

#1: Protein CRISPR-Cas


Mass: 89891.680 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: hot spring metagenome / Source: (gene. exp.) metagenome (others) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: RNA chain RNA (116-MER)


Mass: 39356.418 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: hot spring metagenome / Source: (synth.) metagenome (others)
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: CRISPR-Cas complex / Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: metagenome (others)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 35 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.1_3865: / Classification: refinement
EM software
IDNameVersionCategory
2RELION3image acquisition
8PHENIXmodel refinement
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 469157 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0027944
ELECTRON MICROSCOPYf_angle_d0.49211290
ELECTRON MICROSCOPYf_dihedral_angle_d21.8592086
ELECTRON MICROSCOPYf_chiral_restr0.0321301
ELECTRON MICROSCOPYf_plane_restr0.0041042

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